Supplemental materials and methods

Plants, virus, and inoculation of plants

WClMV-RC was previously reported by Namba and colleagues (Nakabayashi et al., 2002) and was kindly provided by Prof. Namba from the University of Tokyo, Japan. Broad bean (Vicia faba cv. Kawachi-issun and Wase) and pea (Pisum sativum, PI 118501, PI 226564, PI 347295, PI 378159) plants (Andrade et al., 2007) were used in this study. The infectious plasmid clone of WClMV-RC constructed in this study was used to biolistically inoculate broad bean as described previously (Andrade et al., 2007). Infected leaves, which contain the recovered virus from the plasmid, were used to mechanically inoculate pea test plants. Test plants were maintained in a growth chamber at 24°C with 16 h of light and 8 h of dark. The green fluorescent protein (GFP) signal in infected plants was observed with an epifluorescence microscope (SZX-12, Olympus) and recorded with a CCD camera (VB-6010; Keyence, Osaka, Japan).

Cloning and sequencing the WClMV-RC genome

Total RNA was isolated from broad bean leaves infected with WClMV-RC using the TRIzol reagent (Invitrogen Carlsbad, CA, USA). The viral genome cDNAs were synthesized using a cloned AMV reverse transcriptase (Invitrogen) and amplified by polymerase chain reaction (PCR) using KOD plus DNA polymerase (Toyobo, Osaka, Japan). The WClMV genome was divided into four fragments, nucleotide numbers 1-761, 756-2033 (EH), 2028-4209 (HH), and 4204-5843 (HB), according to the nucleotide sequence of the WClMV genome (Nakabayashi et al., 2002). Each cDNA of the four fragments was amplified by PCR with primer pairs, fusionF (5¢-catttggagagggaaaacaagacgaaacg-3¢) and EcoBamR (5¢-cgctacggatcctttgaattcatgggagtaggaac-3¢), EcoF (5¢-gcccatgaattcaaacaactggagtggct-3¢) and HinBamR (5¢-cgtcaggatccggaagcttggagtagtcgtcaaac-3¢), HindF (5¢-ggatcgccaagcttcctcaaggctacatcg-3¢) and HindR (5¢-gcctgaaagctttaattgtggggttggag-3¢), or HindF2 (5¢-cggtcaaagctttcgcccaattcaaaag-3¢) and BamR (5¢-gagccgtactggatccttttttttttttttttttttctgaaattttattaaacagaaagccacac-3¢). In addition, the Cauliflower mosaic virus 35S promoter fragment in an infectious clone of Clover yellow vein virus (Takahashi et al., 1997) was obtained by PCR with BclSalF (5¢-ctcagctgatcagtcgaccttctagagatccgtc-3¢) and fusionR (5¢-cgtttcgtcttgttttccctctccaaatgaaatgaac-3¢). The 35S promoter fragment was fused with the first fragment (1–761) of the WClMV genome with PCR using the primer pair BclSalF and EcoBamR. The fused fragment was named BE. These four cDNA fragments, BE, EH, HH, and HB, which partially overlapped each other and covered the full length of the WClMV-RC genome, were inserted into the pGEM-Teasy plasmid vector (Promega, Madison, WI, USA) and cloned into Escherichia coli strain XL10-Gold (Stratagene, La Jolla, CA, USA). The cloned cDNA fragments were sequenced using an ABI PRISM 310 Genetic Analyzer (Applied Biosystems, Forster, CA, USA).

Construction of the infectious plasmid cDNA clone of WClMV

First, all restriction enzyme sites of the polylinker, except for the BamHI site, on the plasmid vector pBluescript II Sk(-) (Stratagene) were removed to create pBSII/BamHI. The plasmid clone containing BE was digested with restriction enzymes BclI and BamHI, and the BE fragment was size-fractionated on a slab agarose gel. The purified BE fragment was inserted into pBSII/BamHI at the BamHI site. Then the EH fragment digested with EcoRI and BamHI was inserted into pBSII/BamHI containing BE. The HB fragment digested with HindIII and BamHI was inserted into pBSII/BamHI containing BE and EH. Finally, the HH fragment digested with HindIII was inserted into pBSII/BamHI containing BE, EH, and HB. This plasmid contained the full-length cDNA of the WClMV genome following the 35S promoter and was named pWClMV-RCi.

Determination of the transcriptional initiation site of a subgenomic RNA for capsid protein (CP) translation

The initiation site of subgenomic RNA for CP translation was determined using the Gene RacerTM Kit for full-length, RNA ligase-mediated rapid amplification of the 5¢- and 3¢-ends (RLM-RACE, Invitrogen) according to the manufacturer’s manual.

Construction of the WClMV-RCi vector

The WClMV-RCi vector was constructed based on the infectious clone pWClMV-RCi. A partial cDNA, with nucleotide numbers 4472–5166 and a multi-cloning site (MCS) composed of AccIII, SmaI, NheI, SpeI, and XhoI restriction enzyme sites at the 3¢-terminal was made by PCR with primers W-Kp (5¢-atagggcgaattgggtaccatcctcgcgt-3¢) and W-ASNSX (5¢-gcaccttgagctcgagactagtgctagccccgggtccggacattggatttcaattag-3¢). Both the PCR fragment and pBluescritp II SK(-) plasmid were digested with KpnI and XhoI, and the digested PCR fragment was inserted into the pBluescript II SK(-) plasmid at the KpnI-XhoI site and cloned (pBSII/KpnI-MCS). Next, partial WClMV cDNA fragments 50, 75, and 100 bp of the upstream region from the transcription initiation site of the subgenomic RNA for CP translation were amplified by PCR with primer pairs pro50bp (5¢-aagtacctcgagcctcccaaatctca-3¢) and CP-Pst (5¢-atcccccgggctgcaggcatga-3¢) for the cDNA fragment of nucleotide numbers 5555–6069, pro75bp (5¢-cataagttctcgaggcgacaacccggct-3¢) and CP-Pst for 5530–6069, and pro100bp (5¢-cataagttctcgagggagcttctgttg-3¢) and CP-Pst for 5505–6069. These three PCR fragments and pBSII/Kpn-MCS were digested with XhoI and PstI, inserted into the digested pBSII/Kpn-MCS, and cloned (pBSII/Kpn-MCS-Promoter-Pst). The region from the KpnI site to the PstI site of pWClMV-RCi was replaced with that of the pBSII/Kpn-MCS-Promoter-Pst to obtain the WClMV-RCi vectors pWClMV-RCi-50, -75, and -100.

The WClMV-RCi vectors expressing GFP in infected plants were constructed as follows. GFP cDNA with attached AccIII sites at both terminals was amplified by PCR with primers GFP-head (5¢-cctgac tccggaagtaaaggagaagaact-3¢) and GFP-tail (5¢-cgactccggattcatttgtatagttcatc-3¢), using the Clover yellow vein virus (ClYVV) vector pClYVV/C3-S65T (Sato et al., 2003) as a GFP template, and inserted into pWClMV-RCi plasmids at the AccIII sites.

The WClMV-RCi vectors expressing the PDS RNA sequence in pea were prepared. The 470 nt of the partial nucleotide sequence of the PDS gene with attached AccIII sites at both terminals was amplified by PCR with primers PDS-head (5¢-gtgcaatccggacatcaaccctgatgaa-3¢) and PDS-tail (5¢-ctgcagtccggatcgtttagctcaattg-3¢) using cDNA from the total RNA extract of pea PI 226564 as a template for the PDS sequence, and inserted into the pWClMV-RCi-100 plasmid at AccIII sites. The WClMV vectors possessing the PDS sequence in the sense orientation and antisense orientation were designated pWClMV-RCi-PDSs and -PDSas, respectively.

Northern blot hybridization with a DIG labeled cRNA probe

Northern blotting was performed according to a previous study (Yambao et al., 2008). WClMV and PDS cDNA fragments following the T7 promoter were prepared by PCR with primer pairs PDSnorth-head (5¢-ttgaggctcaagatggtgtttctg-3¢) and PDSnorth-tailPDS (5¢-aattctaatacgactcactataggggggattgccatccaaaaaggcc-3¢) for the PDS probe, and CPnorth-head (5¢-cttgcctctcgcttgttgggatctg-3¢) and CPnorth-tail (5¢-aattctaatacgactcactatagggagaaagccacacatattaagtgaa-3¢) for the WClMV probe. The DIG-labeled cRNA probe was transcribed from the PCR fragments.

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